Three-dimensional bioreactors

ABSTRACT

The present invention relates to the design, fabrication and applications of three-dimensional (3D) bioreactor for cell expansion and cell secreted substance production. The bioreactors have relatively low levels of potentially cytotoxic compounds, can be coated with substituted or unsubstituted poly(p-xylene) type coatings and can also be separately formed from liquid crystal photopolymerizable monomers.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of the filing date of U.S.Provisional Application 62/735,531 filed on Sep. 24, 2018, the teachingsof which are incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the design, fabrication andapplications of three-dimensional (3D) bioreactors for cell expansionand cell secreted substance production. The invention includes methodsof reducing or preventing of cytotoxic leaching of 3D bioreactors orother components where fabricated via in situ photo-polymerization based3D printing techniques. The methods include the combination of 3Dprinting material with relatively high photo-polymerization efficiency,post-printing UV curing, post-printing heat/vacuum extraction, andpost-printing coatings with substituted or unsubstituted poly(p-xylene)type material.

BACKGROUND

Cancer immunotherapy, representing the most recent phase ofbiotechnology revolution in medicine, is the use of a patient's ownimmune system to treat cancer. A recent successful case is the chimericantigen receptor (CAR) T-cell based cancer treatment. A typical CART-cell therapy process is one in which T lymphocytes are collected froma patient's own blood are genetically engineered to produce a specialreceptor CAR on their surface so that the T cells are able to recognizeand attack cancer cells. The engineered CAR T cells are grown in thelaboratory and expanded to billions of numbers and then injected back tothe patient to kill cancer cells.

With the successful of CAR T-cell therapy, the next question is how tomake it safer and cost-efficient. The current T-cell engineering processis still generally based on the use of magnetic beads to incubatetogether with T-cells. The magnetic beads are coated with CD3 and CD28on their surface to act as the antigens to activate the T-cells so theycan proliferate. The micro-beads suspended in cell culture medium withT-cells provide a relatively large surface area for T-cells to contactand temporarily bind to CD3 and CD28 and then activate. After T-cellsare grown to a certain density, the T-cells and magnetic beads are movedinto a relatively large bioreactor such as the GE's WAVE bioreactor tocontinue the process of stimulation and expansion. The process isrepeated multiple times to grow relatively large numbers of T-cells. Atharvesting, the T cells have to be separated from the beads using amagnetic separator. Accordingly, the current T-cell expansion processgenerally relies upon magnetic beads, multi-stage processing, and manualinteractions, which is not cost-effective. In addition, it is an opensystem, which can easily introduce contaminations and make it relativelymore expensive to meet good manufacturing process (GMP) requirements.

Accordingly, a need remains for methods and devices to improve cellularexpansion, and in particular T-cell expansion, by offering improvedbioreactor designs, cost-effective fabrication techniques, and improvedbioreactor operating capability in order to achieve clinical applicationdose requirements.

Another pressing need is a device to provide efficient expansion ofadherent cells such as the stem cells. With the recent development ofstem cell technology and regenerative medicine, the number of stemcell-based therapies has increased significantly. Stem cells have thepotential to cure many human diseases because they are not yetspecialized, and can differentiate into many types of cells for tissuerepair and regeneration. Large-scale cell expansion is one of thebottlenecks in stem cell based therapy and tissue regeneration.Typically, there are 10⁹ cells in one gram of tissue. However, thenumber of replicating cells harvested from a donor is extremely low(˜10⁵ cells), which necessitates approximately 10,000-fold cellexpansion for clinical applications. The conventional 2D planar T-flaskmethod for cell culture is relatively difficult to scale-up. The manualprocess using the T-flask is labor-intensive, susceptible tocontamination, and requires high cost to meet cGMP cell manufacturingstandards. Commercially available 3D cell expansion devices based onstacked plates, microcarriers, and hollow fibers also have severallimitations, including: limited scalability, lack of critical processcontrol, high shear stress, large nutrition gradient, and complexdownstream processing. In the present disclosure, we describe thedesign, fabrication, and applications of a device as a 3D bioreactor forcell expansion and production of cell secreted substances.

SUMMARY

A 3D bioreactor for growth of cells comprising a plurality of voidshaving a surface area for cell expansion, said plurality of voids havinga diameter D, a plurality of pore openings between said voids having adiameter d, such that D>d and wherein: (a) 90% or more of said voidshave a selected void volume (V) that does not vary by more than+/−10.0%; and (b) 90% or more of said pore openings between said voidshave a value of d that does not vary by more than +/−10.0%; and coatingsaid bioreactor with substituted or unsubstituted poly(p-xylylene).

A 3D bioreactor for growth of cells comprising a plurality of voidshaving a surface area for cell expansion, said plurality of voids havinga diameter D, a plurality of pore openings between said voids having adiameter d, such that D>d and wherein: (a) 90% or more of said voidshave a selected void volume (V) that does not vary by more than+/−10.0%; and (b) 90% or more of said pore openings between said voidshave a value of d that does not vary by more than +/−10.0%;

wherein said bioreactor comprises the polymerization product of thefollowing monomer:

wherein X and Y comprise polymerizable groups that are polymerized byfree-radical polymerization, R₂ is a bulky organic group having a bulkgreater than R₁ and R₃, wherein R₂ is adapted to provide sufficientsteric hindrance to achieve a nematic state at room temperature.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a section view of the 3D bioreactor fixed-bed.

FIG. 1a illustrates a unit negative model of the bioreactor that showsthe overlapping of the neighborhood spheres.

FIG. 1b illustrates a unit negative model with each sphere surrounded by12 identical neighborhood spheres.

FIG. 1c illustrates a 3D bioreactor fixed-bed geometry showing aninterconnected void system.

FIG. 1d illustrates a 3D bioreactor fixed-bed geometry incross-sectional view.

FIG. 1e illustrates in 2D view the identified spherical voids of a 3Dbioreactor, and their overlapping areas to form interconnected poresbetween the spherical voids.

FIG. 2 illustrates a 3D bioreactor fixed-bed positioned in a housingwith inlet and outlet for fluid perfusion.

FIG. 3 illustrates a typical 3D bioreactor perfusion system.

FIGS. 4a, 4b, 4c and 4d show flow rate profiles through a 3D bioreactor.

FIG. 4e indicates the scale of flow rate through a 3D bioreactor.

FIGS. 5a and 5b illustrate the distribution of surface shear stress in a3D bioreactor.

FIG. 5c indicates a scale of shear stress in unit Pa.

FIG. 6a illustrates the pressure drop (gradient) along the flowdirection in a cylindrical 3D bioreactor.

FIG. 6b indicates a scale of pressure.

FIG. 7a illustrates a 3D bioreactor fixed-bed generated by FDM 3Dprinting.

FIG. 7b illustrates the 3D bioreactor fixed-bed together with abioreactor chamber.

FIG. 7c illustrates the inlet and outlet of a 3D bioreactor.

FIG. 7d illustrates an assembled 3D bioreactor.

FIG. 7e illustrates a 3D bioreactor fixed-bed generated by SLA 3Dprinting.

FIG. 7f illustrates a 3D bioreactor fixed bed generated by DLP 3Dprinting.

FIG. 8 illustrates two fluid distributors placed at the inlet and outletof the 3D bioreactor to approach a laminar flow.

FIG. 9 illustrates a flow rate profile through the 3D bioreactor whenusing the fluid distributor.

FIGS. 10a and 10b illustrate the formation of a 3D bioreactor by thealternative porogen-leaching method.

FIG. 11 illustrates immobilization of antibodies on the 3D bioreactor'sspherical void volume for binding and activation of T-cells.

FIG. 12 illustrates a bead-free, closed loop perfusion based 3Dbioreactor for T-cell activation and expansion.

FIG. 13 illustrates fluorescence intensity of avidin and streptavidincoatings at different concentrations. The fluorescent-labeled avidin andstreptavidin were used to show the concentration of avidin andstreptavidin coated on the bioreactor surface.

FIG. 14 illustrates fluorescence intensity of the biotin coating onavidin or streptavidin. The fluorescent-labeled biotin was used to showthe concentration of biotin bound onto the avidin or streptavidin.

FIG. 15 illustrates T-cell growth (after beads activation) incubated(static) with antibody-coated magnetic beads and bioreactor matrixversus a bioreactor matrix without an antibody coating.

FIG. 16 illustrates T-Cell activation and expansion incubated (static)with beads, bioreactor matrix with antibody coating and bioreactormatrix without antibody coating, respectively.

FIG. 17 illustrates T-cell activation and expansion in a 3D bioreactorwith continuous perfusion.

FIG. 18 illustrates a preferred post-printing protocol for the 3Dbioreactor herein.

FIG. 19 shows the comparative results of a parylene C coated 3Dbioreactor with different lengths of UV/ozone treatment.

FIG. 20 shows cell expansion human derived adipose stem cells (hADSC) ona parylene C coated 3D bioreactor with UV/ozone treatment.

FIG. 21 shows fluorescence microscopic images showing increasing numberof living ADSC cells as a function of time in the 3D bioreactor.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present disclosure relates to a perfusion-based scalable bioreactordesign and with corresponding operating capability to achieve T-cellexpansion for immunotherapy purposes or stem cell expansion forregenerative medicine. The activation and expansion of T-cells or stemcells from a patient, after for example, gene-modification, provides atherapeutic T-cell or stem cell product that can be infused back to thepatient and uses patient's own immune system in a manner thatselectively targets and kills the patient's tumor cells or uses thepatient's own regenerative cells for curing aging related diseases.

Reference to a bioreactor herein refers to the disclosed 3D reactor inwhich biological and/or biochemical processes can be implemented underselected environmental and operating conditions. This includes controlof one or more of the following: geometry/size of the voids,interconnected pore size between the voids and total number of voidsincluded (determining the overall dimension of the bioreactor). Inaddition, one may selective control surface coatings, flowcharacteristics through the voids within the bioreactor, pH,temperature, pressure, oxygen, nutrient supply, and/or waste removal.Clinical dosage requirements are reference to the ability to provide adose of 10⁹ cells or greater.

The 3D bioreactor's preferred fixed-bed 10 is generally illustrated incut-away view in FIG. 1, which shows an example of a preferred packedand spherical void structure and their interconnected pores between thespherical voids. More specifically, the bioreactor includes a continuousinterconnected 3D surface area 12 that provides for the ability for thecells to bind in the present of antibodies and also defines within thebioreactor a plurality of interconnected non-random voids 14 which asillustrated are preferably of spherical shape with internal concavesurfaces to maximize the surface to volume ratio. A void is understoodas an open space of some defined volume. By reference to non-random itshould be understood that one can now identify a targeted or selectednumber of voids in the 3D bioreactor that results in an actual repeatingvoid size and/or geometry of a desired tolerance.

By reference to a continuous surface, it is understood that theexpanding cells can readily migrate from one surface area location intoanother within the 3D bioreactor, and the surface does not include anyrandom interruptions, such as random breaks in the surface or randomgaps of 0.1 mm or more. Preferably, 50% or more of the surface areawithin the 3D bioreactor for cell expansion is a continuous surface,more preferably, 60% or more, 70% or more, 80% or more, 90% or more, 95%or more or 99% or more of the surface area within the 3D bioreactor iscontinuous.

In addition, the bioreactor fixed-bed 10 includes non-randominterconnecting pore openings 16 as between the voids. Again, referenceto non-random should be understood that one can now identify a targetedor selected number of pores for the voids, of a selected pore diameter,that results in an actual number of pores having pore diameters of adesired tolerance. The bioreactor as illustrated in cut-away view alsoultimately defines a layer of non-random voids (see arrow “L”) and itmay be appreciated that the multiple layers of the bioreactor may thenallow for identification of a plurality of such non-random voids withina column (see arrow “C”).

The bioreactor may be made of biocompatible or bio-inert polymericmaterials such as polystyrene, polycarbonate,acrylonitrile-butadiene-styrene (ABS), polylactic acid (PLA),polycaprolactone (PCL) used in FDM (fused deposition modeling) 3Dprinting technology. Reference to biocompatible or bio-inert should beunderstood as a material that is non-toxic to the culturing cells. Inaddition, the polymeric materials for the 3D bioreactor are preferablyselected from those polymers that at not susceptible to hydrolysisduring cell cultivation, such that the amount of hydrolysis does notexceed 5.0% by weight of the polymeric material present, more preferablyit does not exceed 2.5% by weight, and most preferably does not exceed1.0% by weight. The bioreactor may also be made of biocompatiblematerials (e.g., poly(methyl methacrylate) or PMMA, etc.) used in SLA(stereolithography) and DLP (digital light processing) 3D printingtechnologies.

The bioreactor herein may also be preferably made from biocompatible,liquid crystalline, photopolymerizable monomers described in U.S. Pat.Nos. 7,041,234; 7,094,360; 7,098,359; 7,108,801; 7,147,800; and7,238,831, which are hereby incorporated by reference. Accordingly, themonomers herein that may also be employed to form the 3D bioreactorherein may be described as having the following structure:

In the above, X and Y comprise polymerizable groups, which may bepolymerized by free radical polymerization or by nucleophilic addition,including but not necessarily limited to Michael addition. Examplesinclude unsaturated carbon-carbon bonds and epoxy end groups. Preferredend groups therefore may include substituted and unsubstituted alkenylester groups comprising an unsaturated carbon-carbon bond (e.g.—OC(O)—CH═CH₂ or —OC(O)—C(CH₃)═CH₂). R₂ is a bulky organic group havinga bulk greater than R₁ and R₃, wherein R₂ is adapted to providesufficient steric hindrance to achieve a nematic state at roomtemperature while suppressing crystallinity of the liquid crystallinemonomer. Accordingly, R₁ and R₃ are selected from groups less bulky thanR₂. Suitable R₂ groups include but are not necessarily limited tot-butyl groups, isopropyl groups, phenyl groups, and secondary butylgroups. Particularly preferred R₂ groups include t-butyl groups. R₁ andR₃ are less bulky than R₂ and preferably selected from hydrogen atomsand methyl groups. Blends of different monomers of this type may also beused to minimize starting monomer viscosity and polymerization shrinkageand to maximize polymerization conversion to produce minimal residualmonomer and to optimize final polymer mechanical properties. It iscontemplated that starting zero shear monomer viscosity would be in therange of 5 Poise to 300 Poise at temperatures of 25° C. to 70° C.,polymerization conversion would be in the range of at least 75% andhigher, more preferably greater than or equal to at least 90%. Also, theas formed polymer preferably has the following mechanical strengthcharacteristics: (1) flexural modulus at room temperature of at least1500 MPa, and in the range of 1500 MPa to 2500 MPa; (2) flexuralstrength at room temperature of at least 70 MPa and more preferably inthe range of 70 MPa to 100 MPa. Moreover, the polymerization shrinkageat less than or equal to 5%, more preferably in the range of 0.5% to4.0%. In addition the liquid crystalline monomer blends of the structureillustrated above may be mixed with photoinitiator and thermal initiatorsystems and viscosity modifiers while still maintaining the liquidcrystalline state.

It is preferable that the material used to fabricate the bioreactor isnot degradable in aqueous medium and can provide a mechanically stablestructure to tolerate aqueous medium flow during cell expansion. It ispreferable that the material and manufacturing process can result asolid and smooth interconnected surface area for monolayer cellexpansion. By reference to a solid surface, it should be understood thatthe surface is such that it will reduce or prevent penetration orembedding by the culturing cells, which typically have a diameter ofabout 20 microns to 100 microns. Preferably, the 3D bioreactor herein isone that has a surface that has a surface roughness value (Ra), which isreference to the arithmetic average of the absolute values of theprofile height deviations from the mean line, recorded within anevaluation length. Accordingly, it is contemplated herein that Ra of the3D bioreactor surface will have a value of less than or equal to 20 μm,more preferably, less than or equal to 5 μm.

The 3D bioreactor herein is also preferably one that is formed frommaterial that indicates a Shore D Hardness of at least 10, or in therange of 10-95, and more preferably in the range of 45-95. In suchregard, it is also worth noting that the 3D bioreactor herein is onethat does not make use of a hydrogel type structure, which may beunderstood as a hydrophilic type polymeric structure, that includes someamount of crosslinking, and which absorbs significant amounts of water(e.g., 10-40% by weight). It is also worth noting that the 3D bioreactorherein is one that preferably does not make use of collagen, alginate,fibrin and other polymers that cells can easily digest and undergoremodeling.

Furthermore, the 3D bioreactor herein is preferably one that is madefrom materials that have a Tensile Modulus of at least 0.01 GPa. Morepreferably, the Tensile Modulus has a value that is in the range of 0.01GPa to 20.0 GPa, at 0.01 GPa increments. Even more preferably, theTensile Modulus for the material for the 3D bioreactor is in the rangeof 0.01 GPa to 10.0 GPa or 1.0 GPa to 10 GPa. For example, with respectto the earlier referenced polymeric materials suitable for manufactureof the 3D bioreactor herein, polystyrene indicates a Tensile Modulus ofabout 3.0 GPa, polycarbonate at about 2.6 GPa, ABS at about 2.3 GPa, PLAat about 3.5 GPa, PCL at about 1.2 GPa, and PMMA at about 3.0 GPa.

The 3D bioreactor design herein with such preferred regular geometriccharacteristics and continuous surface area is preferably fabricated byadditive manufacturing technologies, such as FDM, selective lasersintering (SLS), stereolithography (SLA), digital light processing (DLP)3D printing technologies, etc., according to computer generated designsmade available by, e.g., a SolidWorks™ computer-aided design (CAD)program.

By way of preferred example, the process utilizing SolidWorks™ to createthe 3D bioreactor design is described below. A computer model for thebioreactor negative is initially created. More specifically, what maytherefore be described as a 3D bioreactor negative was created, e.g.,using packed 6.0 mm diameter spheres that overlap to create 1.0 mmdiameter connecting pores between spheres. Of course, other possibledimensions are contemplated within the broad context of this disclosure.

The spheres are preferably organized in a hexagonal close packed (HCP)lattice to create an efficiently (or tightly) packed geometry thatresults in each sphere surrounded by 12 neighborhood spheres. A unitcell of this exemplary geometry is shown in FIG. 1 a. More specifically,in FIG. 1a there is a unit cell of the HCP lattice where the top threespheres are displayed as translucent to show the 6 radial overlappingareas between the neighborhood spheres. The pores are formed at theseoverlapping areas. Preferably, the maximum number of pores is 12 tooptimize packing. The minimum pore number is 2 in order to allow mediumperfusion through the voids of the 3D bioreactor. Accordingly, at least90.0% to 100% of the voids present in the 3D bioreactor have at least 2pore openings per void. More preferably, at least 90.0% to 100% of thevoids in the 3D bioreactor have 8-12 pore openings per void. In oneparticularly preferred embodiment, at least 90.0% to 100% of the voidsin the 3D bioreactor have 12 pore openings per void.

In FIG. 1 b, all spheres of the unit are illustrated. The bioreactorgeometry is then preferably created by reversing the negative model tocreate the positive model comprising an interconnected spherical voidsystem shown in FIG. 1 c. Moreover, in FIG. 1d one can see the 3Dbioreactor again in cross-sectional view providing another illustrationof the interconnected voids shown in cut-away view at 14 with regulargeometric characteristics (substantially the same control of void volumeas described above) and the corresponding interconnected pore openings16.

In the preferred regular geometric 3D bioreactor described above, onecan identify a relationship as between the void diameter andinterconnected pore diameter. Attention is directed to FIG. 1 e. Forthis preferred geometry, Spherical Void 1 is represented by a solidcircle, diameter is D (indicated by the arrows). Diameter “D” maytherefore be understood as the longest distance between any two pointson the internal void surface. Spherical Void 2 is represented by a dashcircle and would also have diameter D (not shown). Spherical Void 2 isone of the 12 of neighborhood voids of Spherical Void 1. Due to theoverlap between the neighborhood voids, it forms interconnected poresbetween the spherical voids, with the diameter of “d” as also indicatedby the generally horizontal arrow. Diameter “d” may therefore beunderstood as the longest distance between any two points at the poreopening. The total 3D spherical surface area of the void isSA_(void)=4×π×(D/2)². The surface area between A and B, calledS_(cap)=π×D×h, where

$h = {\frac{D - \sqrt{D^{2} - d^{2}}}{2}.}$

The useful void surface for a given void in the 3D bioreactor would beSA_(u)=SA_(void)−[12×S_(cap)].

The smaller the void diameter D, the larger the number of voids can bepacked into a set 3D space (volume), and therefore results largeroverall cell binding surface. However, to minimize or prevent cellaggregation to block the perfusion, the minimal diameter of the pores ispreferred d=0.2 mm for this geometry. The diameter of the pores d mayfall in the range of 0.2 mm to 10 mm and more preferably 0.2 mm to 2.0mm. Most preferably, d≥0.5 mm and falls in in the range of 0.5 mm to 2.0mm.

If D=0.40 mm or less, the computed SA_(u) is less than 0 when d=0.2 mm,which leads to an impossible structure therefore, D has to be >0.4 mmfor this 3D bioreactor geometry. However, D can have a value between 0.4mm to 100.0 mm, more preferably, 0.4 mm to 50.0 mm, and also in therange of 0.4 mm to 25.0 mm. One particularly preferred value of D fallsin the range of 2.0 mm to 10.0 mm. Spherical voids with a relativelylarge value of D may reduce the objective of increasing cell culturesurface area as much as possible within a same bioreactor volume.Accordingly, for the preferred geometry illustrated in FIG. 1 e, D>0.4mm (the diameter of the void) and d>0.20 mm (the diameter of the pores).It is also worth noting that with respect to any selected value ofdiameter D for the voids in the range of 0.4 mm to 100.0, and anyselected value of diameter d for the pores in the range of 0.2 mm to10.0 mm, the value of D is such that it is greater than the value of d(D>d).

It can now be appreciated that the 3D bioreactor herein can becharacterized with respect to its non-random characteristics.Preferably, all of the voids within the 3D bioreactor are such that theyhave substantially the same volume to achieve the most efficient 3Dspace packing and offer the largest corresponding continuous surfacearea. With respect to the total number of interconnected voids presentin any given 3D bioreactor, preferably, 90.0% or more of such voids, oreven 95.0% or more of such voids, or even 99.0% to 100% of such voidshave a void volume (V) whose tolerance is such that it does not vary bymore than +/−10.0%, or +/−5.0%, or +/−2.5% or +/−1.0%, or +/−0.5% or+/−0.1%. It should be noted that while the voids in FIG. 1 are shown asgenerally spherical, other voids geometries are contemplated. Thediameter of voids are chosen to minimize or avoid cell aggregation andto provide maximum useful surface area for cell culturing.

Another non-random characteristic of the 3D bioreactor herein are thepore openings between the voids, having a diameter d (see again FIG. 1e). Similar to the above, 90.0% or more of the pore openings, or even95.0% or more of the pore openings, or even 99.0% to 100% of the poreopenings between the voids, indicate a value of d whose tolerance doesnot vary more than +/−10.%, or +/−5.0%, or +/−2.5% or +/−1.0%, or+/−0.5% or +/−0.1%.

It can therefore now by appreciated that the 3D bioreactor herein forgrowth of non-adherent cells comprises a surface area for cell binding,a plurality of voids having a diameter D (the longest distance betweenany two points on the internal void surface), a plurality of poreopenings between said voids having a diameter d (the longest distancebetween any two points at the pore opening), where D>d. In addition, 90%or more of the voids have a void volume (V) that does not vary by morethan +/−10.0%, and 90% or more of the pore openings have a value of dthat does not vary by more than +/−10.0%.

In addition, the 3D bioreactor herein for expansion of non-adherentcells like T-cells can include a first plurality of voids having adiameter D₁, a plurality of pore openings between said first pluralityof voids having a diameter d₁, wherein D₁>d₁, where 90% or more of thefirst plurality of voids have a void volume (V₁) with a tolerance thatdoes not vary by more than +/−10.0%. Such 3D bioreactor may also have asecond plurality of voids having a diameter D₂, a plurality of poreopenings between said second plurality of voids having a diameter d₂wherein D₂>d₂, wherein 90% of the second plurality of voids have a voidvolume (V₂) with a tolerance that does not vary by more than +/−10.0%.The values of V₁ and V₂ are different and outside of their tolerancevariations. Stated another way, the value of V₁, including its toleranceof +/−10.0% and the value of V₂, including its tolerance of +/−10.0%,are different, or [V₁+/−10.0%]≠[V₂+/−10.0%].

The radius of curvature (Rc) of the surface within the voids istherefore preferably 1/0.5 (D), or 1/0.2 mm=5 mm⁻¹ or lower. Preferably,Rc may have a value of 0.2 mm⁻¹ to 1.0 mm⁻¹, which corresponds to avalue of D of 10.0 mm to 2.0 mm. A high curvature (large Rc) surfaceprovides a significantly different environment than the typicalmonolayer 2D culture, which may also induce cell phenotype changes.

Cells are preferably bound on the interconnected spherical void surfacesof the 3D bioreactor. Such 3D structure is preferably scalable and isable to provide a relatively high surface to volume ratio for relativelylarge cell expansion with a relatively small footprint cell expansionbioreactor. The surface area-to-volume ratio is also preferablydetermined by the diameter of the spherical voids. The smaller is thediameter, the higher is the surface area-to-volume ratio. Preferably,the voids provide a relatively “flat” surface (i.e., low radius ofcurvature≤1.0 mm⁻¹) for cells having a size of 5 μm to 100 μm and alsoto reduce or avoid cell aggregation. In addition, as alluded to above,cell aggregation is also reduced or avoided by controlling the diameterd of the interconnected pores, which diameter is preferably at least 500μm, but as noted, any size greater than 200 μm.

The bioreactor fixed-bed 10 may therefore preferably serve as asingle-use 3D bioreactor as further illustrated in FIG. 2. Morespecifically, the bioreactor 10 may be positioned in a housing 18 andthen placed in the inlet and outlet compartment 20 for which inflow andoutflow of fluid may be provided. Preferably, the bioreactor 10, housing18, and the inlet and outlet compartment 20 can be fabricated as asingle component using Additive Manufacturing technology. As shown inFIG. 3, the bioreactor 10 in housing 18 and inlet and outlet compartment20 may become part of an overall 3D bioreactor system for MSCsexpansion. More specifically, the 3D bioreactor is preferably positionedwithin a perfusion system which delivers a cell culture medium andoxygen through the 3D bioreactor for promoting cell growth. Multiplepassage cell expansion methods used in 2D T-flask can also be directlyapplied to the 3D bioreactor except a 3D bioreactor has the cell culturearea equivalent to 10 s, 100 s, or 1000 s of T-flasks.

As may now be appreciated, the 3D bioreactor herein offers a relativelylarge surface-to-volume ratio depending upon the diameter of theinterconnected voids. By way of example, a conventional roller bottledefining a cylinder of 5 cm diameter and 15 cm height, provides a cellgrowth surface area of 236 cm². If the same volume is used to enclosethe 3D bioreactor herein with 2.0 mm diameter interconnected voids, atotal of 44,968 spherical voids can be packed into the space, which canprovide a matrix with about 5,648 cm² surface area, an almost 24-foldlarger than the roller bottle surface area.

At least one unique feature of the 3D bioreactor herein in comparisonwith hollow-fiber or microcarrier-based bioreactors is the ability toprovide a large interconnected continuous surface instead of fragmentedsurfaces. Continuous surfaces within the 3D bioreactor herein aretherefore contemplated to enable cells to more freely migrate from onearea to another. Using the perfusion system shown in FIG. 3, it iscontemplated that one can readily create a gradient of nutrition or cellsignals inside the bioreactor to induce cell migration.

In conjunction with the preferred 3D printing technology noted hereinfor preparation of the 3D bioreactor, computational fluid dynamics (CFD)can now be used to simulate the medium flow inside the bioreactor andestimate the flow rate and shear stress at any location inside the 3Dinterconnected surface, and allow for optimization to improve the cellculture environment. More specifically, CFD was employed to simulate theflow characteristics through the 3D interconnected voids of thebioreactor herein and to estimate the distribution of: (1) flowvelocity; (2) pressure drop; and (3) wall shear stress. It may beappreciated that the latter parameter, shear stress, is important forcell expansion. A reduction in shear stress can reduce or prevent shearinduced cell differentiation.

A small-scale (to increase computer simulation speed) cylindrical 3Dbioreactor with a diameter of 17.5 mm, height of 5.83 mm, void diameterof 2 mm, and pore diameter of 0.5 mm was used in the simulationsreported below. In this case, the diameter (Φ=17.5 mm) to height (H=5.83mm) ratio of the bioreactor is 3:1 (FIG. 1d ), which is a preferableratio to reduce the gradient of nutrition and oxygen between the inletand outlet of the bioreactor. Based on the cell density available on thefixed-bed spherical surface the oxygen and nutrition consumption rateswere estimated, and how often the cell culture media needed to bereplaced (i.e., the volume flow rate) was determined. An overall linearflow rate of 38.5 μm/sec was assumed in this simulation. Using 38.5μm/sec rate laminar flow as the input to the 3D bioreactor, the CFDresults are shown in FIGS. 4-6.

FIGS. 4 a, 4 b, 4 c and 4 d show the flow velocity profile throughoutthe small-scale cylindrical 3D bioreactor. FIG. 4e indicates the scaleof flow rate. More specifically, FIG. 4a indicates the flow ratedistribution viewed from the side of the bioreactor. The flow passeseach spherical void through the pores along the flow direction. Thewhite/gray areas in the figures are the solid regions between thespherical voids with no fluid flow. By comparing with the coloredvelocity scale bar in FIG. 4 e, FIG. 4a indicates that the flow rate atthe pores along the flow direction achieve the maximum flow rate of 200μm/s to 240 μm/s. In contrast, the flow rates near the spherical surfacereduce to a minimum of 0.06 μm/s to 19.0 μm/s, which will significantlyreduce the flow caused shear stress to cells reside on the sphericalsurface.

FIG. 4b indicates the velocity profile viewed from the top of thebioreactor through a center cross-section of the 3D structure. Again,the image shows that the maximum rates are at each center of the poresof the spherical voids along the flow direction. This maximum rate isagain in the range of 200 μm/s to 240 μm/s. The flow rate near sphericalsurface is again low and has a value of 0.06 μm/s to 19.0 μm/s.

FIG. 4c indicates the velocity profile of an individual sphere voidshowing flow passing through the radial interconnected pores. FIG. 4ctherefore provides a useful illustration of the flow distribution insidea spherical void. The high flow rate is at the central empty space of avoid where there are no cells and is at a level of 200 μm/s to 240 μm/s.The cells reside on the concaved void surface where the flow rate isreduced and where the flow rate is again at a level of 0.06 μm/s to 19.0μm/s. This unique structure can therefore shield cells from exposure torelatively high flow stress. This is another distinct advantage of the3D bioreactor described herein over, e.g., micro-carrier based reactors,where cells are grown on the outside surface surfaces of 300 μm to 400μm diameter microbeads with convex spherical surfaces that are suspendedin a cell culture medium and stirred in a bioreactor to delivernutrition and oxygen to the cells. Cells residing on such convexspherical surfaces can be exposed to relatively large shear stress to0.1 Pa, which is known to affect cellular morphology, permeability, andgene expression. FIG. 4d indicates the flow trajectory through the sidepores along the flow direction, indicating that the 3D bioreactor hereinprovides a relatively uniform flow pattern to provide nutrients andoxygen throughout.

Accordingly, the maximum linear flow rate computed inside the preferred3D bioreactor is 200 μm/s to 240 μm/s which occurs at the 0.5 mmdiameter interconnected pores between 2.0 mm diameter voids along theflow direction. As shown in FIGS. 4a-4e while the flow is preferentiallyin the central direction along the flow, there is still flow (˜19.0μm/sec) near the spherical surface to allow nutritional supply to thecells residing on the spherical surface. Therefore, it is contemplatedthat the cells are able to reside anywhere throughout the structure andthrive in any location because nutrients can be supplied both throughflow convection and diffusion throughout the 3D bioreactor structure.

FIGS. 5a and 5b show the distribution of surface shear stress throughoutthe cylindrical 3D bioreactor described above as well as on a singlespherical void surface. FIG. 5c indicates the scale of shear stress inunits of Pa. The highest shear stress was observed on the edges of theinterconnected pores. This is due to the higher flow rates at theselocations. However, the majority of the useful spherical surface areawithin the bioreactor indicates a shear stress of less than 3×10⁻⁴ Pa,which may be understood as 90% or more of the surface area of thebioreactor. This provides for cell proliferation, without shear induceddifferentiation. In addition, even the maximum shear stress of 4.0×10⁻³Pa, is believed to be lower than the average shear stress that cellsexperience when cultured in hollow fiber based bioreactors, wavebioreactors, and micro-carrier based bioreactors. Therefore, the 3Dbioreactor herein is contemplated to provide a relatively lower shearstress environment for cell growth in comparison to existing cellexpansion bioreactors. See, e.g., Large-Scale Industrialized CellExpansion: Producing The Critical Raw Material For BiofabricationProcesses, A. Kumar and B. Starly, Biofabrication 7(4):044103 (2015).

FIG. 6a illustrates the pressure drop along the flow direction frombottom to the top of the cylindrical 3D bioreactor described above. FIG.6b provides the applicable scale of pressure. The figure indicates thatthe overall pressure drop between the inlet and outlet of the bioreactoris less than or equal to 1.0 Pa. The pressure drop may therefore fall inthe range of 0.1 Pa up to 1.0 Pa. In other words, cells near the inletand outlet of the bioreactor will not experience significant differencesin pressure. The low gradient of pressure suggests that such design willalso produce a small gradient (or difference) in nutrition/metabolitesconcentrations between the inlet and outlet of the bioreactor. The lowgradient is due to the design of the bioreactor such that the diameter Φis larger than the height H while the total bioreactor volume remainsthe same. This is superior to the hollow fiber bioreactor. It isdifficult to fabricate a hollow fiber bioreactor with Φ>H ratio toreduce the gradient of nutrition/metabolites between the inlet andoutlet of the bioreactor.

A comparison was also made for the same total volume cylindrical 3Dbioreactor with different aspect ratios (i.e. Φ:H ratio, Φ: overalldiameter of the bioreactor fixed-bed, H: overall height of thebioreactor fixed-bed). See FIG. 1 d. As shown in Table 1, for the samevolume flow rate (volume flow rate=cross area of flow×linear velocity),the linear velocity increases significantly for a bioreactor with a lowΦ:H ratio. The increase of linear velocity also increases the surfaceshear stress, pressure drop, as well as the gradient ofnutrition/metabolites concentrations between the inlet and outlet, whichwould have an unfavorable effect for cell expansion. The disclosedfixed-bed 3D bioreactor is therefore preferably designed into a Φ:Hratio structure, e.g., a Φ:H ratio in the range of greater than 1:1 andup to 100:1. Preferably, the Φ:H ratio is greater than 1:1 and up to10:1.

TABLE 1 Flow Rate Comparison For 3D Bioreactor With Different AspectRatios Ratio Diameter (Φ) Height (H) Flow Rate # (Φ:H) (cm) (cm)(μm/sec) 1 3:1 10.5 3.5 38.5 2 1:1 7.5 7.5 75.4 3 1:3 5 15 169.8

FIG. 7a illustrates a 3D bioreactor fixed-bed part generated by FDM 3Dprinting with interconnected 6 mm diameter voids and 1 mm interconnectedpores. This 3D bioreactor was printed with ABS filament. The diameter(Φ) and height (H) of this particular 3D bioreactor is 4.28 cm and 1.43cm respectively. Accordingly the Φ:H ratio is 3:1. There are about 134interconnected open-voids included in the fixed-bed. The totalinterconnected continuous spherical surface area SA_(u) for cellculturing is about 152 cm². The inlet and outlet wall and fluiddistributor 22 at the inlet and outlet (FIG. 8) provides an additional88 cm² surface area for cell culturing. In other words, there is about240 cm² total useful surface area in the 3D bioreactor for cellattachment. The fluid distributor can improve the laminar flow throughthe bioreactor. The fluid distributor is optional if the Reynolds numberis <2100 or in the range of greater than 0 up to and not including 2100.

FIG. 7b shows that the fixed-bed of the 3D bioreactor was solvent boundinto a bioreactor chamber. This will seal the gaps between the fixed-bedand the chamber wall, which will force the perfusion cell culture mediumto pass through the interconnected pores instead of through those gaps.Preferably, the fixed-bed and chamber is printed together as anintegrated part to increase the manufacturing efficiency. FIG. 7cillustrates the inlet and outlet of the bioreactor. They are designedgeometrically to promote a laminar flow through the fixed-bed. The inletof the bioreactor optionally contains a built-in rotation gear, whichmay be coupled to a stepper motor to control the rotation of thebioreactor for uniform cell seeding (see below). The integratedbioreactor is shown in FIG. 7d and is able to connect to ⅛ inch tubingto conduct the fluid flow. Alternatively, the inlet and outlet can bemade for repeated usage, where only the inside bioreactor fixed bed isdisposable. Also shown in FIG. 7e is a relatively smaller-size 3Dbioreactor fixed bed produced by DLA 3D printing having a 3.0 mm voidand a 0.5 mm pore. FIG. 7f is a relatively larger-size 3D bioreactorfixed bed using DLP 3D printing having the same-diameter 3.0 mm void anda 0.5 mm pore. When scaling up the bioreactor, the internal structure ofthe bioreactor is maintained. Therefore, the perfusion volume flow ratecan be scaled up proportionally while keeping the local linear flow ratethe same. In this way, minimum process change is required during thescale-up process which can reduce the process development costs.

It should next be noted that the fluid distributor 22 (FIG. 8) ispreferably such that it will improve the flow uniformity through the 3Dbioreactor. The design of the inlet, outlet, and fluid distributor alsopreferably takes into consideration the following: (1) improve the flowuniformity through the 3D bioreactor; (2) minimization of thedead-volume 24 at inlet and outlet to reduce the overall priming volumeof the bioreactor; and (3) preventing bubble collection inside thebioreactor. FIG. 9 shows the flow velocity profile throughout the 3Dbioreactor based on CFD simulation by using the fluid distributor. Theuse of the fluid distributor (FIG. 8) improved the uniformity of theflow. The maximum flow rate (around 30 μm/s) and the minimum flow rate(around 10 μm/s) are relatively close to each other and serve to promoteuniform laminar flow (i.e. flow of fluid in relatively parallel layers).A relatively uniform flow rate everywhere in the bioreactor will alsoprovide smaller differences of shear stress to cells residing atdifferent locations in the bioreactor.

The 3D bioreactor can be fabricated by other additive manufacturingtechnologies such as selective laser sintering (SLS), stereolithography(SLA), Digital Light Processing (DLP), and etc. FIGS. 7 e, 7 f.

In addition to preparing the 3D bioreactor herein via additivemanufacturing or 3D printing, it is contemplated that the 3D bioreactormay be prepared by the traditional porogen-leaching method to provide aninterconnected cell culture surface. FIGS. 10a and 10b shows a 3Dbioreactor utilizing a porogen-leaching methodology. This is referenceto combining porogen and polymer in a mold, followed by leaching out ofthe porogen to generate pores. The 3D bioreactor in FIG. 10a starts withthe step of tightly packing 4.0 mm water-soluble spherical sugar beads(as porogen) in a cylindrical stainless mold by shaking, tapping, andpressing the beads, so that the beads are in contact. The gaps betweenthe beads are filled with acetone containing 5.0% by weight deionizedwater. This is followed by evaporation the acetone and water undervacuum chamber overnight. The gaps between the beads are then filledwith a low viscous polymerizable vinyl monomer such as styrene togetherwith polymerization initiators such as benzoyl ortert-butylperoxybenzoate. The styrene monomer will then polymerize toform polystyrene. The sample remained at 90° C. for 8-12 hours, and thenwas heated to 115° C. for an additional 3 hours and removed from theoven to provide what is illustrated in FIG. 10 a. The sugar beads werethen leached out while submerged in an ultrasound water bath to leavethe polystyrene 3D bioreactor fixed-bed with interconnected voids. SeeFIG. 10 b. The 3D bioreactor is then extracted with methanol for threedays to remove any residual styrene monomer. However, the porogenleaching method not only has a complex manufacturing process, but alsois difficult to achieve exact reproducible structures since the packingof porogen beads is a random process.

For the 3D printed bioreactor (FIG. 7d ) using ABS or PMMA, thehydrophobic internal surfaces of the bioreactor are preferably modifiedto allow for cell adherence. Polydopamine was used as a primer coatingto the bioreactor surfaces so that other proteins can be easily adhereto the bioreactor surface via the polydopamine coating. It therefore canbe noted that the polydopamine primer coating can be combined with othercoatings such as peptides, collagen, fibronectin, laminin, multiple cellextracellular matrix proteins or selected antibodies that are requiredby particular cell types.

Incubation of the bioreactor surface in a 0.25 mg/mL dopamine dissolvedin 10 mM Tris buffer (pH=8.5 at 25° C.) for a period of about 18 hours,resulted in an effective polydopamine layer for the subsequent proteincoating. After polydopamine is deposited on the bioreactor surface,other proteins can then bind with functional ligands via Michaeladdition and/or Schiff base reactions. The ligand molecules thereforeinclude nucleophilic functional groups, such as amine and thiolfunctional groups.

It can also be appreciated that when the bioreactor herein is coatedwith substituted or unsubstituted poly(p-xylene), polydopamine may besimilarly applied over the substituted or unsubstituted poly(p-xylene)coating to similarly allow for cell adherence. Again, the polydopaminemay then provide a coating surface so that other proteins can adhere viathe polydopamine coating over the poly(p-xylene) coating. Such bindingis again contemplated to rely upon protein binding via functionalligands via Michael addition and/or Schiff base reactions and the ligandmolecules can again include nucleophilic functional groups, such asamine and thiol functional groups. It can therefore more generally beunderstood that the polydopamine coating is such that it is capable ofbinding additional layer or layers for cell culturing via the use offunctionalized ligands.

It should now be appreciated from all of the above that one of theadditional features of the 3D bioreactor disclosed herein is that onemay now design a 3D bioreactor, with particular geometric and voidvolume requirements, and corresponding available surface arearequirements, and be able to achieve (i.e., during fabrication ormanufacturing) such targets with relatively minimal variation. Forexample, one may now identify a design requirement for a 3D bioreactorwherein the one or more internal voids are to have a targeted voidvolume “V_(t)”, and the 3D bioreactor itself is to have a targetedoverall surface area for cell culturing “SA_(t)”. Accordingly, one maynow form such 3D bioreactor wherein the one or more internal voids havean actual void volume “V_(a)” that is within +/−10.0% of V_(t), or morepreferably, +/−5.0% of V_(t). Similarly, the actual surface area forcell culturing SA_(a) is within +/−10.0% of SA_(t), or more preferably+/−5.0% of SA_(t). Moreover, one may also identify for the internalsurface within the targeted voids a targeted geometry for fabricationsuch as a targeted radius of curvature “Rc_(t)” and then in fabricationthe actual radius of curvature “Rc_(a)” of the void internal surface cannow be achieved that is within +/−5% of Rc_(t).

Post Printing Process

The 3D bioreactor herein is now preferably subjected to additionalpost-printing procedures. Such procedures are designed to provide theability to reduce or prevent leaching of any residual monomers and/orphotoinitiators that may otherwise have been employed for 3D bioreactorprinting. Such may particularly be the case when the additivemanufacturing procedures involve stereolithography (SLA) or digitallight processing (DLP) which involves curing (polymerization) of aliquid resin. Attention is therefore directed to FIG. 18 whichsummarizes a preferred post-printing protocol. As illustrated, the 3Dbioreactor herein produced by additive manufacturing that initiallyrelies upon photo-polymerization (light-induced polymerization) is nowpreferably post-cured, preferably by additional exposure to UV light.Such additional exposure to UV light is contemplated to increase theunderlying polymerization during printing and/or to reduce the level ofrelatively low molecular weight material, such as un-polymerizedmonomeric material. In addition, one may now follow such post-curingwith treatment of heat and/or vacuum to further reduce or remove suchrelatively low molecular weight material. For example, one may apply avacuum of 10⁻³ Ton or lower at temperatures from ambient to 125° C. Itis therefore contemplated that by use of post-curing, alone or incombination with vacuum and/or heating, one may reduce the level ofrelatively low MW material (MW of less than or equal to 500) to lessthan or equal to 2.0% by weight, more preferably to a level of 0.01% byweight to 1.50% by weight, and even more preferably 0.01% by weight to0.75% by weight.

In addition, it is contemplated herein that for any given bioreactorproduced herein, it may now be preferably subject to a coating procedureto similarly reduce and/or eliminate the leaching of the aforementionedrelatively low molecular weight compounds that are otherwise toxic toliving cells. Such coatings may also be selectively functionalized topromote adherent cell attachment and growth. Preferably, the coatingprocedure relies upon the use of parylene monomers, e.g.,[2.2]paracyclophanes, that may be functionalized with identified R₁, R₂,R₃ and R₄ groups according to the following general reaction scheme,wherein the indicated polymerization is promoted by exposure to heat(˜550° C.) under vacuum to provide a substituted or unsubstitutedpoly(p-xylylene) polymer structure. It should be appreciated that in thescheme below, the start of polymerization is initiated by a ring openingat elevated temperature in the gas phase prior to deposition on the 3Dbioreactor which is preferably maintained at relatively lowertemperature (e.g., ≤100° C.):

In the above, when one of the R groups per repeat unit “m” and/or repeatunit “n” is chlorine, and the other R group is a hydrogen, the aboverepresents the polymerization of parylene C. It is a USP Class VI andISO-10993-6 certified biocompatible material. The values of “m” and “n”of the identified repeating units are such that molecular weight valuesare relatively high, such as ˜500,000. It is therefore contemplated thatthe use of the parylene monomers and ensuing polymeric coatings are suchthat one may now coat the entire 3D bioreactor herein with animpermeable film. The film may preferably have a thickness between 200Angstroms to 100.0 μm. It may be appreciated that R₁, R₂, R₃, and R₄ maybe selected from hydrogen, a halogen (—Cl or —Br) as well as otherfunctional groups such as amines (—NH₂), aliphatic aldehydes (—CHO),carboxylic acid functionality (—COOH), hydroxyl (—OH) or carboxylatefunctionality as in —C(O)CF₃. One may also initially coat with a firstlayer of parylene C followed by a coating of a different parylene, e.g.,wherein R₁, R₂, R₃, and R₄ may then be selected from an amines (—NH₂)and/or aldehyde (—CHO) functionality. Accordingly, one may providepolymeric coatings for the 3D bioreactor herein, wherein the coatingcomprises a plurality of layers, each with its own particular anddifferent chemical composition (i.e. the identity of at least one of R₁,R₂, R₃, and R₄ are different between at least two of the layers).

It is further contemplated that the 3D bioreactor with a functionalizedcoating of an aldehyde can undergo reaction with, e.g., antibodyproteins (e.g. anti-CD3/28) with end flexible tethers that are aminoterminated (or other organic terminal group) of an oligoethylene oxide(OEG) of different lengths. In other words, the use of poly(ethyleneoxide) type tethers that include functional terminal groups such as anamine group, as in:

where the value of n may be in the range of 1-200, and which may thenbind to the functionalized parylene coating on the 3D bioreactor hereinas follows, where one binding reaction site is illustrated and where itshould be appreciated that multiple binding reactions may take placedepending upon regulation of the reaction parameters (e.g. temperatureand time to increase binding reaction yield):

As may therefore be appreciated, in the above, one may vary the surfacemobility of the cell stimulating protein to enable better binding withthe targeted, cell surface receptors and minimize non-specific proteinor other macromolecule physical absorption from the cell culture media.

In another contemplated embodiment, biotin, with an end functionalizedtether, can be bound to the functionalized parylene coating in a firststep. In a second step the biotin end group can be complexed with avidinor streptavidin. In a third step the biotin-strepavidin complex is thenbound to the biotin functionalized stimulatory antibody to complete thefunctional surface appropriate for cell stimulation.

The present invention advances further on the use of the abovereferenced 3D bioreactor herein, to provide for T-cell expansion asapplied for immunotherapy purposes. Reference is made to FIG. 11, whichillustrates immobilization of antibodies on the bioreactor's sphericalvoid volume surface for binding and activation of T-cells. Morespecifically, for the 3D bioreactor, one can now immobilize antibodies(biotin conjugated sphere-shaped proteins) such as anti-CD3 andanti-CD28 antibodies on the bioreactor surface via thebiotin-avidin/streptavidin binding mechanism. Avidin or streptavidin arepre-coated on the bioreactor surface through the prime polydopaminecoating as discussed above. More specifically, after a polydopaminecoating is applied to the surface of the 3D bioreactor herein, which asnoted already can include a poly(p-xylene) coating, one may attach atetrameric protein (protein with quaternary structure of four subunits)such as avidin or streptavidin, which have affinity for biotin.Accordingly, it may be appreciated that the polydopamine coatingprovides the ability to make the bioreactor surface relatively morehydrophilic and for attachment of proteins and antibodies where bycontrast, the poly(p-xylene) coating serves to block monomer leachingand to reduce or avoid cytotoxicity. Accordingly, proteins withquaternary structure (avidin or streptavidin) may be relied upon toimmobilize biotinylated antibodies, for example, anti-CD3 antibody ontothe bioreactor surface. A biotinylated antibody is reference to covalentattachment of biotin to the antibody. When T-cells flow through thebioreactor, they will bind and activate via the T-cell surface receptor,for example CD3, via the CD3, anti CD3 antibody binding. This is thenfollowed by exposing the activated T-cells to a perfusion mediacontaining a signaling molecule to provide T-cell expansion. Thesignaling molecule may preferably include a cytokine signaling moleculesuch as interleukin-2 IL-2). It should be noted, however, that in thebroad context of the present invention, it is contemplated that one mayexclude the need for the use of a polydopamine coating and immobilizethe antibody (e.g. the anti-CD3 antibody) directly on the surface of the3D reactor, avidin/streptavidin directly on the bioreactor surface tobind biotinylated antibodies.

Using the 3D bioreactor herein, a closed-loop perfusion-based system forT-cell expansion is now possible as illustrated in FIG. 12. Morespecifically, FIG. 12 illustrates a bead-free, closed-loop perfusionbased 3D bioreactor for T-cell activation and expansion. The perfusionsystem preferably contains a cell-safe peristaltic pump and amedium/cell reservoir, which is able to add media to dilute cell densityfor more effective cell expansion or exchange media to maintain thenutrient concentration during the expansion process. The mediumreservoir may also serve as an oxygenator with gas infusion and stirringor shaking mechanism to mix the nutrients and oxygen. The suspendedT-cells in the medium perfused through the 3D bioreactor have manychances of contacting CD3 and CD28 antibodies on the spherical surfacesand thus to be activated. The immobilized CD3 and CD 28 on the largesurface is expected to provide equivalent stimulation to T-cells as themagnetic beads used in the current T-cell expansion system. Without thebeads, the processes will be simplified significantly. In addition, thisclosed-loop system facilitates automation and cost-effective cGMP cellmanufacturing.

Table 2 lists the dimensions, culture surface area, number of magneticbeads with equivalent total surface area, expected medium volume, etc.of three different sized bioreactors. PMMA, an FDA approved implantablebiocompatible material, was used to fabricate the bioreactor using a DLP3D printer. Table 2 also lists the approximate material cost toconstruct the identified 3D bioreactors.

TABLE 2 Dimension and Cost of Fabricating Different Sized 3D BioreactorsUsing SLA/DLP 3D Printing PBMC Seeding Capacity 2.5 × 10^(7α) 2.2 × 10⁸2.2 × 10⁹ Bioreactor diameter (cm) 2.1 4.2 9.0 Bioreactor height (cm)0.7 1.4 3.0 Surface area (cm²) 29 250 2500 Area equivalent to # of 7.5 ×10⁷ 6.5 × 10⁸ 6.5 × 10⁹ beads Medium volume (mL) 2 14 136 Build materialvolume 0.78 5.4 53.6 (mL) Material cost^(β) $0.20 $1.34 $13.4 ^(α)basedon 3:1 beads:cells ratio ^(β)matrix only, does not include bioreactor'sinlet and outlet; also assume 3 mm diameter hollow sphere and 0.5 mmpore size

Coating of Avidin (or Streptavidin) on the Bioreactor Surface

To mimic the Miltenyi MACSiBead system, avidin (streptavidin) and biotinbinding mechanism was employed to immobilize anti-CD2, CD3 and CD28antibodies on the polydopamine coated bioreactor surface. First,different concentrations of fluorescence-labeled avidin and streptavidinwere tested (FIGS. 13) and 100 μg/mL of avidin or 30 μg/mL ofstreptavidin was found to achieve a relatively high avidin orstreptavidin coating density on the bioreactor surface. FIG. 13 showsflorescence intensity of avidin and streptavidin coating at differenceconcentrations. The fluorescent-labeled avidin/streptavidin were used totest the concentration of avidin or streptavidin that can be bound on tothe polydopamine prime coating. A preferred procedure ofavidin/streptavidin coating is described as follows. The coatingconcentration of avidin or streptavidin can be further optimized:

1) The bioreactor surfaces are first coated by polydopamine herein.2) Dissolve avidin or streptavidin in TRIS buffer (pH 8.5) to preparethe coating solution, with the avidin or streptavidin concentration of100 μg/mL or 30 μg/mL, respectively. Immerse the bioreactor into thecoating solution for 12 hours, while gentle shaking in the dark.3) Wash the scaffolds thoroughly with phosphate-buffered-saline (PBS).Then leave the scaffolds in PBS, ready for coating with biotinylatedantibodies.

A comparison was then run with respect to different concentrations offluorescence-labeled biotin as a second layer coating to bind to theavidin or streptavidin base layer (FIG. 14). 1.0 μg/mL of biotin wasidentified as successfully binding to the immobilized avidin orstreptavidin. See FIG. 14, which identifies fluorescence intensity(proportional to biotin concentrations) of the biotin coating on avidinor streptavidin. After confirming that biotin can successfully bind tothe bioreactor surfaces coated with avidin or streptavidin, biotinylatedanti-CD2, CD3, and CD 28 antibodies were applied onto the bioreactor forT cell activation and expansion.

To preferably apply the antibody coating on to the 2.1 cm diameterscaffolds (i.e., equivalent to about 7.5×10⁷ beads total surfaces):

1) Remove the bioreactor scaffolds from the BPS2) Remove as much PBS from the scaffolds as possible, but do not let thescaffold dry.3) Combine the 200 μL of 100 μg/mL CD2-Biotin, 200 μL of 100 μg/mLCD2-Biotin, and 200 μL of 100 μg/mL CD28-Biotin in a 15-mL tube, add 1.4mL of antibody labeling buffer (PBS without Ca2+ and Mg2+, pH=7.2 plus0.5% heat-inactivated fetal bovine serum and 2 mM EDTA), and mix well tomake a total 2 mL biotinylated antibodies, with the concentration ofeach antibody being 10 μg/mL.4) Add 2 mL of the mixed antibodies to cover a bioreactor matrix(without inlet and outlet, FIG. 7e ) in a 12 well plate, pipette up anddown to mix.5) Incubate in matrix in the dark in the refrigerator for 2 hours undergentle shaking at a temperature of 2° C. to 8° C.6) Remove the unbound antibodies from the scaffold thoroughly with PBSwash.

The coating procedures using polydopamine, avidin/streptavidin, andbiotinylated antibodies can be extended to coat the internal surface ofan intact bioreactor (that is, the bioreactor matrix plus the inlet andoutlet).

Comparison of T Cells Incubated with Antibody-Coated Bioreactor Matrixand Magnetic Beads for T-cell Expansion

An antibody-coated matrix (without inlet and outlet) was compared withthe Miltenyi MACSiBead™ system. The magnetic beads (already coated withstreptavidin) were coated with biotinylated anti-CD2, CD3, and CD28antibodies according to the manufacturer's protocol, which is similar tothe coating procedure described above. The relatively small 2.1 cmdiameter by 0.7 cm height bioreactor (FIG. 7e ), listed in Table 1, wasused. The bioreactor matrix fits in a well of a 12-well plate so thatone can easily compare the performance of beads and matrix. Threebioreactor matrices were coated with avidin or streptavidin first, andthen the biotinylated CD2, CD3, CD28 antibodies using the preferredprocedures described above. The same concentrations of antibodies wereused to coat the bioreactor matrix and the MACSiBeads. One bioreactormatrix with streptavidin coating was not further coated with antibodies(the negative control)

Human peripheral blood CD3+ Pan T cells (ReachBio Research Labs) werefirst activated and expanded (7-day) using the MACSiBeads according tothe manufacturer's protocol and then removed from the magneticparticles. Then 4.5×10⁶ T cells were added to four wells of a 12-wellplate, respectively. Four wells, each filled with 3 mL of culture medium(RPMI 1640 supplemented with 10% fetal bovine serum and 20 IU/mL ofhuman IL2), contained either 1) 7.5×10⁶ antibody-coated magnetic beads,2) antibody-coated streptavidin-matrix, 3) antibody-coatedavidin-matrix, and 4) streptavidin-matrix without antibody coating,respectively. Additional medium was added to the well on Day 3. On Day5, the cells were divided into two wells with additional beads andmatrices. The number of T cells in each well, after being dissociatedfrom the magnetic beads or the matrix, were counted on Days 3, 5 and 7.

FIG. 15 shows T-cell growth (after activation) incubated withantibody-coated magnetic beads and matrices versus a matrix without anantibody coating. The trend of T cell growth in antibody coated-matricesis similar to that of antibody-coated beads. However, T cells incubatedwith the matrix without antibodies did not proliferate, probably due tothe lack of continued stimulation. In this experiment, the matrix wasplaced in a well under static condition. Therefore, the results areexpected to be different from the perfusion based bioreactor. Thisexperiment clearly indicate that the antibodies were successfully coatedon the bioreactor's matrix surfaces, and they promoted the T cellgrowth.

Comparison of Peripheral Blood Mononuclear Cells (PBMCs) Incubated withAntibody-Coated Bioreactor Matrix and Magnetic Beads for Both T-CellActivation and Expansion

A similar study to the above was performed with PBMCs instead ofisolated T-cells. Typically, PBMCs, which include T cells and othermononuclear such as B cells, NK cells, monocytes, were collected from apatient and directly used for cell expansion without T-cell isolation.This is because non-T cells will not be activated by CD2, CD3, and CD28,and they will be naturally eliminated after several days withoutactivation. Another difference of this experiment from the experiment inFIG. 15 is the incorporation of the cytokine activation step when usingthe antibody-coated matrix. Briefly, 2 mL of culture medium containingPBMCs at density of 2.5×10⁶ cells/mL were added to three wells of a12-well plate. The three wells contain 1) 7.5×10⁶ antibody-coatedmagnetic beads, 2) antibody-coated streptavidin-matrix, and 3)streptavidin-matrix without antibody coating, respectively. Avidincoated matrix was not used in this experiment as streptavidin has shownsuperior to avidin in the last two experiments. PBMCs were incubated ineach well for two days for activation. Then additional media containingIL2 cytokine were added to the well so that the media has 20 IU/mL ofhuman IL2 to start the expansion phase. The change of the total cellnumber during the 5-day culture is illustrated in FIG. 16. This confirmsthat the T-cells expanded with antibody-coated matrix resulted in more Tcells when compared with antibody coated magnetic beads. This alsoconfirms that the antibody-coated matrix can provide equivalent orbetter T-cell activation and expansion.

Dynamic Perfusion Based T-Cell Expansion

A perfusion-based 3D bioreactor was fabricated as described herein. The3D bioreactor's internal surface was prepared by coating withpolydopamine for 12 hours. The bioreactor internal surface was thencoated with streptavidin for 12 hours. The bioreactor was then incubatedwith 70% ethanol for sterilization. After sterilization, the internalsurface was washed with PBS and coated with an equal mixture of CD2,CD3, and CD28-Biotin conjugates (10 μg/mL concentration for eachantibody) to immobilize antibodies on the bioreactor's internal surfacesusing the procedure described above.

A perfusion circuit was set up as illustrated in FIG. 12. A MasterflexL/S with Cytoflow pump head (Cole-Parmer), which is designed for pumpinglive cells and shear-sensitive fluids, was used for the perfusionsystem. As this application uses a relatively small bioreactor and theperfusion rate is expected to be relatively low, a custom-builtoxygenation unit using gas diffusion into the medium and medium dropletswas employed instead of an oxygenator.

About 20×10⁶ PBMCs were seeded into the primed bioreactor perfusionsystem. The cells were distributed evenly by circulating for 15 minutesat 2 mL/min. During the activation phase, (the perfusion mediumcontaining no cytokine IL2 promoter), the T-cells were perfused at therate of 0.1 mL/min on Day 1 and 0.14 mL/min on Day 2. After two days ofactivation, cell density was determined and media with human IL-2cytokine was added to the system so that the total IL-2 concentrationwas 20 IU/mL. The T-cell expansion phase was carried out for 3 days at aperfusion rate of 0.2 mL/min. The cell density was determined on day 5and the results are shown in FIG. 17. Similar to the static experimentshown in FIGS. 15 and 16, T cells achieved a three-fold of expansionafter activation/promotion.

Ozone/UV Surface Treatment of Parylene C Coated 3D Bioreactor

As noted above, the parylene coatings may be functionalized. By way ofexample, in the case of a parylene C coated bioreactor herein, such wastreated with an ultraviolet/ozone system, which is contemplated toprovide hydroxyl, carbonyl and carboxylate surface groups. Morespecifically, the 3D bioreactor was placed in a closed chamber with UVtransparent glass on one side. A UV lamp (254 nm) is placed above theglass about 1.75″ in distance to yield a 3.2 mW/cm² exposure fluxapplied simultaneously during ozone exposure. The inlet of the closedchamber was connected to a VMUS-4 ozone generator (Azco Industries)through PTFE tubing and the outlet of the closed chamber was connectedto a glass bottle filled with mineral oil, all within a fume hood. Theozone generator was set at 2 L/min inlet flow of air. The output was setto an efficiency of 100% to deliver about 2.5 g of ozone per hours.Parylene C coated bioreactors were ozone treated for 1, 2, 5, 10 and 15minutes, respectively. As can be seen from the Table 3 blow, treatingthe parylene C surface for 10 minutes made the surface as hydrophilic asa polydopamine/fibronectin (PD/FB) treated surface. See U.S. patentapplication Ser. No. 15/585,812 the teachings of which are incorporatedherein by reference.

TABLE 3 Effects Of Coating Procedure On Water Contact Angle SurfaceTreatment Water Contact Angle Parylene C coated bioreactor 73.7 ± 9.5Polydopamine/fibronectin 44.0 ± 9.5 Ozone/UV 1 min 64.7 ± 6.0 Ozone/UV 2min 53.0 ± 5.0 Ozone/UV 5 min 51.0 ± 3.5 Ozone/UV 10 min 41.7 ± 3.1Ozone/UV 15 min 34.7 ± 4.0

FIG. 19 shows that UV/ozone treating the parylene C coating on thebioreactor for 5 min was optimal for cell multiplication of adherentmesenchymal stem cells (MSC) that had been previously seeded on thebioreactor. Increase in fluorescence intensity using a MTT assay is aresult of increasing numbers of cells. The cell multiplication at thisexposure exceeded even that achieved by the polydopamine and fibronectincoated bioreactor for both 4 and 7 days. This indicates that therelatively more complex PD/FB treatment is unnecessary. FIG. 20 showscell expansion of human derived adipose stem cells (hADSC). FIG. 21shows the corresponding fluorescence microscope images showingincreasing number of ADSC cells as a function of time in the 3Dbioreactor.

Sterilization and Packaging

It is contemplated herein that in lieu of gamma radiation sterilizationof the 3D bioreactors herein, that may now contain parylene film typecoatings, sterilization may rely upon perfused chlorine dioxide (ClO₂)gas prior to antibody surface immobilization in the case of non-adherentT-cell expansion. The ClO₂ gas is not contemplated to react with afunctionalized parylene film coating (i.e. —Cl, —CHO, —COOH, —NH₂ or—C(O)CF₃) or the underlying 3D bioreactor polymer substrate.

For example, the 3D bioreactor herein may now be sealed in a polymericfilm which may then be placed in an atmosphere of chlorine dioxide forthe purpose of sterilization. Subsequently, such sterilized 3Dbioreactors would be exposed to a flowing air stream to release anyremaining chlorine dioxide gas. Additional methods of sterilizationinclude steam, ozone, hydrogen peroxide, sulfur dioxide and/or ethyleneoxide sterilization.

1. A 3D bioreactor for growth of cells comprising a plurality of voidshaving a surface area for cell expansion, said plurality of voids havinga diameter D, a plurality of pore openings between said voids having adiameter d, such that D>d and wherein: (a) 90% or more of said voidshave a selected void volume (V) that does not vary by more than+/−10.0%; and (b) 90% or more of said pore openings between said voidshave a value of d that does not vary by more than +/−10.0%; wherein saidbioreactor has a coating of a substituted or unsubstitutedpoly(p-xylylene).
 2. The 3D bioreactor of claim 1 wherein saidbioreactor contains 0.01% by weight to 0.25% by weight of materialhaving a MW of less than or equal to
 500. 3. The 3D bioreactor of claim1 wherein said poly(p-xylylene) has the following structure,

wherein R₁, R₂, R₃, and R₄ are selected from hydrogen, a halogen, amine(—NH₂), aliphatic aldehyde (—CHO), carboxylic acid functionality(—COOH), hydroxyl (—OH) or carboxylate functionality comprising—C(O)CF₃.
 4. The 3D bioreactor of claim 1 wherein said coating has athickness of 200 Angstroms to 100.0 μm.
 5. The 3D bioreactor of claim 1wherein said poly(p-xylylene) coating comprises a plurality of coatinglayers having different chemical composition.
 6. The 3D bioreactor ofclaim 1 wherein polydopamine is applied to said poly(p-xylene coating)wherein said polydopamine is capable of binding additional layer orlayers for cell culturing.
 7. The 3D bioreactor of claim 3 wherein saidsubstituted poly(p-xylene) coating is bonded to a poly(ethylene oxide)which includes a terminally substituted antibody protein.
 8. The 3Dbioreactor of claim 3 wherein at least one of R₁, R₂, R₃, and R₄ areselected from an aldehyde (—CHO) and said 3D bioreactor is bonded to apoly(ethylene oxide) having a terminally substituted antibody protein,via a linkage having the following structure: —CH═N—.
 9. The 3Dbioreactor of claim 6 further including an extracellular matrix proteinattached to said polydopamine coating.
 10. The 3D bioreactor of claim 6further including a tetrameric protein attached to said polydopaminecoating.
 11. The 3D bioreactor of claim 10 further including abiotinylated antibody immobilized on said tetrameric protein.
 12. The 3Dbioreactor of claim 10 wherein said tetrameric protein comprises avidinor streptavidin.
 13. The 3D bioreactor of claim 11 wherein saidbiotinylated antibody comprises anti-CD-3 antibody.
 14. The 3Dbioreactor of claim 11 wherein said biotinylated antibody comprisesanti-CD3 antibody and anti-CD28 antibody.
 15. The 3D bioreactor of claim11 wherein said biotinylated antibody comprises anti-CD3 antibody,anti-CD28 antibody and anti-CD2 antibody.
 16. A method of forming a 3Dbioreactor comprising: forming said 3D bioreactor by additivemanufacturing wherein said 3D bioreactor includes a plurality of voidshaving a surface area for cell expansion, said plurality of voids havinga diameter D, a plurality of pore openings between said voids having adiameter d, such that D>d and wherein: (a) 90% or more of said voidshave a selected void volume (V) that does not vary by more than+/−10.0%; and (b) 90% or more of said pore openings between said voidshave a value of d that does not vary by more than +/−10.0%; and coatingsaid bioreactor with substituted or unsubstituted poly(p-xylene). 17.The method of claim 16 wherein said additive manufacturing comprisesstereolithography (SLA) or digital light processing (DLP).
 18. Themethod of claim 17 wherein said bioreactor, prior to coating withsubstituted or unsubstituted poly(p-xylene), is exposed to UV light. 19.The method of claim 17 wherein said bioreactor, prior to coating withsubstituted or unsubstituted poly(p-xylene), is exposed to vacuum. 20.The method of claim 17 wherein said bioreactor, prior to coating withsubstituted or unsubstituted poly(p-xylene), is exposed to heating. 21.The method of claim 16 wherein said substituted or unsubstitutedpoly(p-xylene) coating is exposed to UV light and ozone.
 22. A 3Dbioreactor for growth of cells comprising a plurality of voids having asurface area for cell expansion, said plurality of voids having adiameter D, a plurality of pore openings between said voids having adiameter d, such that D>d and wherein: (a) 90% or more of said voidshave a selected void volume (V) that does not vary by more than+/−10.0%; and (b) 90% or more of said pore openings between said voidshave a value of d that does not vary by more than +/−10.0%; wherein saidbioreactor comprises the polymerization product of the followingmonomer:

wherein X and Y comprise polymerizable groups that are polymerized byfree-radical polymerization, R₂ is a bulky organic group having a bulkgreater than R₁ and R₃, wherein R₂ is adapted to provide sufficientsteric hindrance to achieve a nematic state at room temperature.